The invention relates to coactivators of transcription and to proteins with protein methyltransferase activity.
The activities of all cells are conducted primarily by the thousands of different types of proteins each cell produces. The blueprint or code for synthesizing each protein is found in a corresponding gene, i.e., each gene encodes the information needed to synthesize a specific protein. Gene xe2x80x9cexpressionxe2x80x9d results in the production of the protein by a stepwise mechanism that includes 1) xe2x80x9ctranscriptionxe2x80x9d of the gene by RNA polymerase to produce a messenger RNA (mRNA) that contains the same protein-encoding information; and 2) xe2x80x9ctranslationxe2x80x9d of the mRNA by ribosomes to produce the protein. Each gene is expressed in specific tissues and at specific times during the life of the organism. Expression of most genes is regulated in response to a variety of signals that arise either outside or inside the organism. This pattern of specific expression for each gene is determined by the xe2x80x9cpromoter regionxe2x80x9d of each gene, which is located adjacent to the protein-encoding region of the gene. Each gene""s promoter contains many xe2x80x9cregulatory elements.xe2x80x9d Each regulatory element serves as a binding site for a specific protein, and the binding of the appropriate protein to a specific regulatory element can cause enhancement or repression of gene expression. Together, the regulatory elements and the proteins that bind to these elements determine the expression pattern for the specific gene.
Hormones represent one of the most important mechanisms for communication between different organs and tissues in multicellular organisms. In mammals, hormones are synthesized in one organ or tissue, and travel through the blood stream to various target organs. By interacting with specific receptor proteins in the target cells, the hormones change the activities of the cell. Frequently the cellular effects of the hormone include changes in the expression of specific genes. The protein products of these genes then carry out the biological actions that result in altered cellular functions.
The effects of one extremely important class of hormones are carried out by a family of related receptor proteins called the nuclear receptors (Evans, R. M. (1988) Science 240:889-895; Tsai, M-J. and B. W. O""Malley (1994) Annu. Rev. Biochem. 63:451-486; Beato, M., et al. (1995) Cell 83:851-857). This family of proteins includes the receptors for all of the steroid hormones, thyroid hormones, vitamin D, and vitamin A, among others. The family also includes a large number of proteins called xe2x80x9corphan receptorsxe2x80x9d because they do not bind any hormone or because the hormone that binds to them is unknown; but they are nevertheless structurally and functionally related to the hormone-binding nuclear receptors. Nuclear receptors are transcriptional regulatory proteins that act by a common mechanism. For those nuclear receptors that do bind hormones, the appropriate hormone must enter the cell and bind to the nuclear receptors, which are located inside the target cells. The activated nuclear receptors bind to specific regulatory elements associated with specific genes that are regulated by these proteins. Binding of the activated nuclear receptors to the regulatory elements helps to recruit RNA polymerase to the promoter of the gene and thereby activates expression of the gene. This mechanism also applies to many of the orphan nuclear receptors.
After nuclear receptors bind to a specific regulatory element in the promoter of the gene, they recruit RNA polymerase to the promoter by a mechanism which involves another group of proteins called coactivators, that are recruited to the promoter by the nuclear receptors (Horwitz, K. B. et al. (1996) Mol. Endrocrinol. 10:1167-1177; Glass, C. K. et al. (1997) Curr. Opin. Cell Biol. 9:222-232). The complex of coactivators helps the receptors to activate gene expression by two different mechanisms: 1) they make the gene more accessible to RNA polymerase by unfolding the xe2x80x9cchromatin.xe2x80x9d Chromatin is composed of the DNA (which contains all the genes) and a large group of DNA-packaging proteins. To unfold chromatin some of the coactivator proteins contain an enzymatic activity known as a histone acetyltransferase (HAT). HAT proteins transfer an acetyl group from acetyl CoA to the major chromatin proteins, which are called xe2x80x9chistones.xe2x80x9d Acetylation of the histones helps to unfold chromatin, thus making the gene and its promoter more accessible to RNA polymerase. 2) The coactivators and the nuclear receptors make direct contact with a complex of proteins called basal transcription factors that are associated with RNA polymerase; this interaction recruits RNA polymerase to the promoter. Once RNA polymerase binds to the promoter, it initiates transcription, i.e., synthesis of mRNA molecules. The final activation of RNA polymerase after it binds to the promoter may also require some intervention by the coactivator proteins, but little is known about the mechanism of these final steps of transcriptional activation.
One specific family of three related coactivator proteins, the xe2x80x9cnuclear receptor coactivatorsxe2x80x9d or xe2x80x9cp160 coactivatorsxe2x80x9d (because their mass is approximately 160 kilodaltons), are required for the gene activation activities of many of the nuclear receptor proteins. The three related nuclear receptor coactivators are GRIP1, SRC-1, and p/CIP; all three proteins also have additional names that are used by some investigators (Onate, S. A. et al. (1995) Science 270:1356-1357; Hong, H. et al. (1996) Proc. Natl. Acad. Sci. USA 93:4948-4952; Voegel, J. J. et al. (1996) EMBO J. 15:3667-3675; Kamei, Y. et al. (1996) Cell 85:403-414; Torchia, J. et al. (1997) Nature 387:677-684; Hong, H. et al. (1997) Mol. Cell. Biol. 17:2735-2744; Chen, H. et al. (1997) Cell 90:569-580; Anzick, S. L. et al. (1997) Science 277:965-968; Li, H. et al. (1997) Proc. Natl. Acad. Sci. USA 94:8479-8484; Takeshita, A. et al. (1997) J. Biol. Chem. 272:27629-27634). These coactivators are recruited directly by the DNA-bound nuclear receptors. The nuclear receptor coactivators, in turn, recruit other coactivators, including CBP (or p300) and p/CAF (Chen, H. et al. 1997). All of these coactivators have been shown to play roles in gene activation by one or both of the two mechanisms mentioned above. Some of them have HAT activities to help unfold chromatin structure (Chen, H. et al. 1997; Spencer, T. E. et al. (1997) Nature 389:194-198), and others have been shown to make direct contact with proteins in the RNA polymerase complex (Chen, H. et al. 1997; Swope, D. L. et al. (1996) J. Biol. Chem. 271:28138-28145). Thus, the discovery and characterization of these coactivators provides a better understanding of the mechanism by which nuclear receptors activate gene transcription.
Histones are known to be methylated as well as acetylated (Annunziato, A. T. et al. (1995) Biochem. 34:2916; Gary J. D. and Clarke, S. (1998) Prog. Nucleic Acids Res. Mol. Biol. 61:65). However, the function of histone methylation is unknown. Methylation of histone H3, is a dynamic process during the lifetime of histone molecules, and newly methylated H3 is preferentially associated with chromatin containing acetylated H4 (Annunziato, A. T. et at. 1995); thus methylation of H3, like acetylation of H4, is associated with active chromatin. In other studies lysine methylation of histones has been found in a variety of organisms; arginine methylation of histones, while not clearly documented in mammals, has been demonstrated in other classes of organisms (Gary and Clarke 1998). In Drosophila cells heat shock treatment causes increased arginine methylation of histone H3, which could be associated with activation of heat shock genes or repression of the other genes (Desrosiers, R. and R. M. Tanguay (1988) J. Biol. Chem. 263:4686).
Proteins can be N-methylated on amino groups of lysines and guanidino groups of arginines or carboxymethylated on aspartate, glutamate, or the protein C-terminus. Recent studies have provided indirect evidence suggesting roles for methylation in a variety of cellular processes such as RNA processing, receptor mediated signaling, and cellular differentiation (Aletta, J. M. et al. (1998) Trends Biochem. Sci.: 23:89; Gary and Clarke 1998). However, for the most part the specific methyltransferases, protein substrates, and specific roles played by methylation in these phenomena have not been identified. Two types of arginine-specific protein methyltransferase activities have been observed, type I and type II. Genes for three mammalian and one yeast type I enzymes, which produce monomethyl and asymmetric dimethylarginine residues previously have been identified (FIG. 1). On the other hand, type II protein arginine methyltransferases produce monomethyl and symmetric dimethylarginine residues. In vitro protein substrates for various protein arginine methyltransferases include histones and proteins involved in RNA metabolism such as hnRNPA1, fibrillarin, and nucleolin (Lin, W-J. et al. (1996) J. Biol. Chem. 271:15034-15044.; Gary, J. D. et al. (1996) J. Biol. Chem. 271:4585; Najbauer, J. et al. (1993) J. Biol. Chem. 268:10501-10509). The arginine residues methylated in many of these proteins are found in glycine-rich sequences, and synthetic peptides mimicking these sequences are good substrates for the same methyltransferases (Najbauer, J. et al. 1993).
The invention relates to a transcriptional coactivator, Coactivator Associated arginine (R) Methyltransferase (CARM1).
One aspect of the invention includes CARM1 cDNA polynucleotides such as (SEQ ID NO: 1). Polynucleotides include those with sequences substantially equivalent to SEQ ID NO: 1, including fragments thereof. Polynucleotides of the present invention also include, but are not limited to, a polynucleotide complementary to the nucleotide sequence of SEQ ID NO: 1.
Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers, i.e. primers for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of antisense DNA or RNA, their chemical analogs and the like. For example, when the expression of an MRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the specific MRNA in the particular cell or tissue RNA using, e.g., in situ hybridization. The invention also includes vectors encoding the polynucleotides of the invention.
The invention also describes the deduced amino acid sequence of the CARM1 protein (SEQ ID NO: 2). The invention also describes isolated CARM1 proteins.
The polypeptides according to the invention can be used in a variety of procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide. The invention describes antibodies that specifically interact with the CARM1 protein or fragments thereof.
The polypeptides of the invention also act as methyltransferases of histones and other proteins and can therefore be used for the study of methylation processes in transcription and to methylate amino acid residues within histones and other proteins.
Methylated proteins produced by the methods of the invention can be used to identify demethylating enzymes. Methylated histones, for example, can be used to screen for demethylating enzymes.
The methods of the present invention further relate to the methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample. Such methods can, for example, be utilized as a prognostic indicator of diseases that involve CARM1, modified forms of CARM1, or altered expression of CARM1.
Methods are also provided for identifying proteins that interact with CARM1 as well as methods for screening of drugs that alter CARM1""s interactions with other proteins.
Another aspect of the invention is to provide methods to screen for molecules that alter CARM1 methyltransferase activity.